Identification of novel polyphenol oxidase inhibitors by enzymatic one-pot synthesis and deconvolution of combinatorial libraries.

The feasibility of enzymatic synthesis of combinatorial libraries using multifunctional starting materials [i.e., 2,4-dihydroxy-N-(2-hydroxyethyl)benzamide, 1; 4-hydroxyphenethyl alcohol, 2; 3,5-dihydroxybenzyl alcohol, 3; and 4-hydroxybenzyl alcohol, 4] with six vinyl esters, in a one-pot reaction, was investigated. Candida antarctica lipase was employed as a biocatalyst. The resulting 24-compound library contained all the expected species with no significant bias toward particular combinations of substrates. As expected, the library contained a substance(s) that showed significant inhibition of polyphenol oxidase, which was used as a model target. The deconvolution was accomplished via resynthesis of ten partial libraries, which were prepared with either an equimolar mixture of the four alcohols and a single vinyl ester, or a single alcohol and equimolar mixture of the activated esters. Analysis of the inhibition pattern observed with these partial libraries suggested that 4-hydroxybenzyl benzoate (4e) should be the most potent inhibitor. This conclusion was confirmed by the preparation and comparison of all 24 components of the initial library. Finally, it was shown that 4e was a competitive inhibitor of polyphenol oxidase, with a K(i) of 40 microM. This value compared favorably with a K(i) of 400 microM, which was determined for parent phenol 4.

Phage display combinatorial libraries of short peptides: ligand selection for protein purification.

A library of heptapeptides displayed on the surface of filamentous phage M13 was evaluated as a potential source of affinity ligands for the purification of Rhizomucor miehei lipase. Two independent selection (biopanning) protocols were employed: the enzyme was either physically adsorbed on polystyrene or chemically immobilized on small magnetic beads. From screening with the polystyrene-adsorbed lipase it was found that there was a rapid enrichment of the library with "doublet" clones i.e. the phage species which carried two consecutive sequences of heptapeptides, whilst no such clones were observed from the screening using lipase attached to magnetic beads. The binding of the best clones to the enzyme was unambiguously confirmed by ELISA. However the synthetic heptapeptide of identical sequence to the best "monomeric" clone did not act as a satisfactory affinity ligand after immobilization on Sepharose. This indicated that the interaction with lipase was due to both the heptapeptide and the presence of a part of the phage coat protein. This conclusion was further verified by immobilizing the whole phage on the surface of magnetic beads and using the resulting conjugate as an affinity adsorbent. The scope of application of this methodology and the possibility of preparing phage-based affinity materials are briefly discussed.

Enantioselectivity of recombinant Rhizomucor miehei lipase in the ring opening of oxazolin-5(4H)-ones.

Enantioselectivity of enzyme catalysis is often rationalized via active site models. These models are constructed on the basis of comparing the enantiomeric excess of product observed in a series of reactions which are conducted with a range of homologous substrates, typically carrying various side chain substitutions. Surprisingly the practical application of these simple but informative 'pocket size' models has been rarely tested in genetic engineering experiments. In this paper we report the construction, purification and enantioselectivity of two recombinant Rhizomucor miehei lipases which were designed to check the validity of such a model in reactions of ring opening of oxazolin-5(4H)-ones.

Alteration of lipase chain length specificity in the hydrolysis of esters by random mutagenesis.

The feasibility of altering the chain length specificity of industrially important Rhizomucor miehei lipase was investigated by randomly mutating Phe94 in the protein groove which is responsible for accommodating the acyl chain of the substrate. The recombinant lipase was initially expressed in E. coli. Individual colonies were selected, grown, and the DNA sequence of the lipase gene determined. Fourteen of the 19 possible mutants were identified and each of these was transformed into Pichia pastoris which expresses the enzyme extracellularly. The yeast was grown and the supernatants assessed in several assays with long and short chain substrates. Based on this preliminary screen, one mutant, Phe94Gly, was selected and purified to homogeneity for further analysis. It was found that the substitution of phenylalanine 94 with glycine led to an enzyme which was about six times less active against resorufin ester but displayed 3-4 times higher activity with short chain substrates such as butyric acid esters. The observed alteration to the enzyme specificity was rationalised using the available 3D structure of the lipase.

Analysis of conformational states of Candida rugosa lipase in solution: implications for mechanism of interfacial activation and separation of open and closed forms.

In this study, an analysis of the transition between the inactive ("closed") and active ("open") conformations of Candida rugosa lipase in solution is performed using irreversible enzyme inhibitors, cyclic saligenin phosphates. It is shown that >90% inhibition of the enzyme activity toward water-soluble substrates (esterolytic activity) can be achieved with as little as 0.3 mol of the inhibitor per mole of enzyme, whereas activity toward emulsified substrates decreases by approximately 20% under the same conditions. It is also shown that short-term exposure of this inhibited enzyme preparation to an interface leads to a significant increase in esterolytic activity, which even exceeds that of the untreated control. These experimental observations suggest that the inhibitors interact predominantly, if not exclusively, with the open form of the enzyme and that any transitions occurring between the two conformers of the enzyme in solution, in the absence of an interface, are extremely slow. This conclusion is verified by separating the open and closed forms of the enzyme by hydrophobic interaction column chromatography on phenyl-sepharose. Fractions enriched with the respective conformations of the enzyme are further purified using gel-permeation chromatography. On the basis of the elution pattern from this step, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the open (active in the absence of interface) form of the lipase is found to be present in solution as a dimer, whereas the closed form appears to be a monomer. The latter form of the enzyme may be activated by up to 60-fold when exposed to triolein.

Regioselectivity of enzymatic glycosylation of 6-O-acyl glycosides in supersaturated solutions.

The regioselectivity of enzymatic transglycosylation of 6-O-acetyl glycosides in supersaturated solutions was investigated using a range of commercially available enzymes, Escherichia coli, barley, and Kluyveromyces spp. beta-galactosidase, green coffee bean alpha-galactosidase, jack bean alpha-mannosidase, rice alpha-glucosidase, and almond beta-glucosidase. It has been shown that 6-O-acetyl glycosides serve as good substrates for these enzymes, which, under the reaction conditions, are "forced" to transfer monosaccharide units to the secondary hydroxyl groups of the acceptors. In a variety of transglycosylations studied the (1-3)-linked disaccharide products were the predominant regioisomers isolated. The selectivity of the reaction varied significantly depending on the acceptor glycosides and the enzyme used. Exquisite specificity was observed in some cases, but in others approximately equal quantities of two disaccharides products were isolated. In the best transfers the yield approached 30%. The methodology described offers a quick and facile route to disaccharides that may be difficult and/or time consuming to make by conventional chemical synthesis.

Purification, molecular cloning, and expression of the gene encoding fatty acid 13-hydroperoxide lyase from guava fruit (Psidium guajava).

Guava fruit was identified as a particularly rich source of 13-hydroperoxide lyase activity. The enzyme proved stable to chromatographic procedures and was purified to homogeneity. Based on gel filtration and gel electrophoresis, the native enzyme appears to be a homotetramer with subunits of 55 kD. Starting with primers based on the peptide sequence, the enzyme was cloned by polymerase chain reaction with 3' and 5' rapid amplification of cDNA ends. The sequence shows approximately 60-70% identity to known 13-hydroperoxide lyases and is classified in cytochrome P450 74B subfamily as CYP74B5. The cDNA was expressed in Escherichia coli (BL21 cells), with optimal enzyme activity obtained in the absence of isopropyl-beta-D-thiogalactopyranoside and delta-aminolevulinic acid. The expressed enzyme metabolized 13(S)-hydroperoxylinolenic acid over 10-fold faster than 13(S)-hydroperoxylinoleic acid and the 9-hydroperoxides of linoleic and linolenic acids. 13(S)-Hydroperoxylinolenic acid was converted to 12-oxododec-9(Z)-enoic acid and 3(Z)-hexenal, as identified by gas chromatography-mass spectrometry. The turnover number with this substrate, with enzyme concentration estimated from the Soret absorbance, was approximately 2000/s, comparable to values reported for the related allene oxide synthases. Distinctive features of the guava 13-hydroperoxide lyase and related cytochrome P450 are discussed.

A novel approach to the recovery of biologically active oligosaccharides from milk using a combination of enzymatic treatment and nanofiltration.

A new easily scalable approach to the recovery of biologically active oligosaccharides from milk has been developed which relies on the combination of enzymatic treatment of defatted milk using beta-galactosidase and nanofiltration. It was shown that enzymatic hydrolysis of lactose significantly improves the efficiency and selectivity of membrane-based separations. With the best membrane, as much as 6.7 g of oligosaccharides (containing very little contaminating lactose) could be obtained from one liter of defatted human milk in just four nanofiltration cycles. The human milk oligosaccharides recovered by this method were shown to inhibit binding of intimin, an adhesion molecule of enteropathogenic Escherichia coli, to epithelial cells in vitro. No significant difference in the oligosaccharide profile between samples prepared by this method and conventional gel-permeation chromatography was found. The developed approach is also suitable for the recovery of substantial quantities of tri- and tetra-saccharides from caprine milk.

Novel enzymatic approach to the synthesis of flavonoid glycosides and their esters.

Flavonoids such as (+)catechin can be efficiently solubilised in supersaturated solutions prepared with donor glycosides, e.g., p-nitrophenyl glycosides, di- and higher oligosaccharides, and poly(ethylene glycol) dimethyl ether in sufficiently high concentration for their efficient enzymatic glycosylation. Under these conditions several glycosidases readily accept (+)catechin as substrate and the target glycosides were prepared in one step in up to 26% yields. The regioselectivity of the reaction depends on the enzyme and substrate combination used; three positions, 5, 7, and 4', in the flavonoid can be glycosylated. The resulting and similar flavonoid glycosides were further modified by regioselective acylation with vinyl esters of arylpropenoic acids using lipases as biocatalyst. The efficiency of acylation was found to diminish in the order of vinyl cinnamate > vinyl ferulate > vinyl coumarate. This work demonstrates the feasibility of assembling complex flavonoid glycoside esters in just two steps by sequential use of commercially available glycosidases and lipases.

Combinatorial approach to flavor analysis. 2. Olfactory investigation of a library of S-methyl thioesters and sensory evaluation of selected components.

The odor characteristics of individual components present in a library comprised of S-methyl thioesters were determined independently by two laboratories using similar but not identical techniques. The odor potency was assessed by values of best estimate-GC-lower amount detected by sniffing (BE-GC-LOADS). For small and medium chain S-methyl thioesters, these values were found to increase from 6 ng for S-methyl thiobutanoate to 90 ng for S-methyl thiostearate. All assessors detected a "green", "floral", or "pineapple" odor for S-methyl thiohexanoate and described thioesters containing a 2-6 carbon chain length as "cheesy". The results of this preliminary analysis were confirmed by a more extensive study of selected compounds, namely S-methyl thioacetate, S-methyl thiopropionate, S-methyl thiobutanoate, and S-methyl thiohexanoate, using a trained panel of 18 subjects. The subjects confirmed the presence of the "green" and "fruity" notes in the odor of S-methyl thiohexanoate. The analysis also revealed a significant difference in the odor of S-methyl thiopropionate relative to that of S-methyl thioacetate and S-methyl thiobutanoate. When "cheesy" characteristics were mentioned, the majority of panelists clearly associated the flavor of S-methyl thiopropionate with Camembert with almost 20% of all the descriptors given referring specifically to this cheese variety as compared to about 2 and 5% in the case of S-methyl thioacetate and thiobutanoate, respectively. Prompted by this observation, two samples of Camembert prepared from unpasteurized and pasteurized milk were analyzed and relatively large amounts of S-methyl thiopropionate were found in the former but not in the latter cheese. The results obtained in the course of this work suggest that the sensory analysis of combinatorial libraries is a useful new approach in the search for new commercial flavors and/or identification of characteristic flavors in foods.

Combinatorial approach to flavor analysis. 1. Preparation and characterization of a S-methyl thioester library.

A new method for the "one-pot" synthesis of S-methyl thioesters has been developed by reacting methyl chlorothiolformate with carboxylic acids. The resulting "flavor library" contained all the intended thioesters and a single major impurity, identified by GC-MS as S, S-dimethyldithiocarbonate. Quantification of individual compounds present in the library was performed by GC analysis using two independent methods of detection, SCD and FID. It was shown that apart from S-methyl thioacetate (0.8 mol %), molar concentrations of other thioesters varied in a relatively narrow range from 4.2 mol % for S-methyl thiopropionate to 14.1 mol % for S-methyl thiohexanoate. In general, medium chain S-methyl thioesters were present in slightly higher molar concentrations than those prepared from short or long chain carboxylic acids. This variation was attributed to partial loss of the most volatile components during extraction and the lower reactivity of higher homologues. The library was used for the characterization of some physicochemical parameters of thioesters. In particular, lipophilicity coefficients (log k(w)) and thioester retention in 10, 20, and 33% triolein (used as a model lipid phase) were determined directly by reverse-phase HPLC and extrapolated from the respective data. This analysis illustrates that substantial information can be generated using a library containing a relatively large number of compounds in effectively the same way as is necessary for the analysis of a single sample.

Bacterial adhesion at synthetic surfaces.

A systematic investigation into the effect of surface chemistry on bacterial adhesion was carried out. In particular, a number of physicochemical factors important in defining the surface at the molecular level were assessed for their effect on the adhesion of Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli. The primary experiments involved the grafting of groups varying in hydrophilicity, hydrophobicity, chain length, and chemical functionality onto glass substrates such that the surfaces were homogeneous and densely packed with functional groups. All of the surfaces were found to be chemically well defined, and their measured surface energies varied from 15 to 41 mJ. m(-2). Protein adsorption experiments were performed with (3)H-labelled bovine serum albumin and cytochrome c prior to bacterial attachment studies. Hydrophilic uncharged surfaces showed the greatest resistance to protein adsorption; however, our studies also showed that the effectiveness of poly(ethyleneoxide) (PEO) polymers was not simply a result of its hydrophilicity and molecular weight alone. The adsorption of the two proteins approximately correlated with short-term cell adhesion, and bacterial attachment for L. monocytogenes and E. coli also correlated with the chemistry of the underlying substrate. However, for S. aureus and S. typhimurium a different pattern of attachment occurred, suggesting a dissimilar mechanism of cell attachment, although high-molecular-weight PEO was still the least-cell-adsorbing surface. The implications of this for in vivo attachment of cells suggest that hydrophilic passivating groups may be the best method for preventing cell adsorption to synthetic substrates provided they can be grafted uniformly and in sufficient density at the surface.

Induction of catalytic activity in proteins by lyophilization in the presence of a transition state analogue.

The induction of catalytic activity in proteins by lyophilization in the presence of a transition state analogue (biomolecular imprinting) has been attempted. It was shown that proteins which were freeze-dried with n-isopropyl-4-nitrobenzyl-amine (a transition state analogue for the reaction of dehydrofluorination of 4-fluoro-4-[p-nitrophenyl] butan-2-one) displayed higher beta-elimination activity as compared to their-non-imprinted counterparts. It was also found that native bovine serum albumin has a high dehydrofluorination activity towards the above substrate with kinetic parameters rather similar to those of a catalytic antibody prepared by Shokat et al. (1989). A comparison of the kinetic parameters determined in this study with those obtained for analogous catalytic antibodies and imprinted polymers was made.

Enzymatic transformations in supersaturated substrate solutions: I. A general study with glycosidases.

The results of an initial study of enzymatic catalysis in metastable supersaturated solutions of carbohydrates are presented. It has been shown that such solutions, formed in the presence of small amounts of water and alcohol as plasticizers, are sufficiently stable under ambient conditions to enable enzymatic transformations of substrates. A partial phase diagram for a system consisting of glucose, water, and (poly)ethylene glycol was constructed to identify the regions which are most suitable for biotransformations. It was confirmed that the glass transition in this system occurred below the reaction temperature at any given composition of the constituent components. Several glycosidases were found to be catalytically active in this medium and the activity of beta-glucosidase from almond was determined at several compositions of the reaction mixture and related to the corresponding regions of the phase diagram. The synthetic utility of the system was illustrated by glucosylation of several alpha,omega-alkyldiols, short-chain polyethylene glycols, and hydroxyalkyl and glyceryl monoacrylates.

Enzymatic transformations in supersaturated substrate solutions: II. Synthesis of disaccharides via transglycosylation.

Enzymatic transglycosylation in supersaturated solutions of substrates was investigated using crude glycosidase preparations from barley, snail, and coffee beans. It was shown that the use of supersaturated glycoside solutions as media for transglycosylation reactions offers considerable advantages over conventional aqueous systems. These advantages include higher yields, more efficient use of the donor glycosides and improved volumetric productivity, especially in the case of poorly water-soluble substrates. The regioselectivity of the glycosylation was not significantly affected by high concentrations of acceptor glycosides. It was also shown that the regioselectivity of transfer could be directed towards secondary hydroxyl groups by the use of methyl 6-O-acetyl-alpha-galactopyranoside as acceptor. The value of these approaches was demonstrated by the synthesis of methyl 3- and 4-O-beta-D-galactopyranosyl-alpha-D-galactopyranosides and methyl 3-O-beta-D-galactopyranosyl-alpha-L-fucopyranoside on a preparative scale.

A novel approach to biotransformations in aqueous-organic two-phase systems: enzymatic synthesis of alkyl beta-[D]-glucosides using microencapsulated beta-glucosidase.

A novel approach to enzymatic biotransformations in aqueous-organic two-phase systems was developed where the aqueous phase was contained within permeable polymeric capsules suspended in organic solvent. Microencapsulated beta-glucosidase, used as a model enzyme, was shown to retain its catalytic activity for a considerable time and was repeatedly used in batch experiments after recharging the microcapsules with solid glucose. The reaction conditions for the synthesis of hexyl beta-[D]-glucopyranoside were optimized with regard to the polymer composition of the microcapsules, pH, and the volume ratio of aqueous to organic phases. The potential for further improvement in the efficiency of the system was demonstrated by designing a bioreactor which incorporated units for product recovery and recycling of the organic solvent. Other advantages of the proposed methodology include facile control over the size and composition of the microcapsules, and mild reaction conditions during their preparation.

The synthesis of sub-micron magnetic particles and their use for preparative purification of proteins.

A novel one-step protocol for the preparation of sub-micron magnetic particles as small as 30 nm in diameter has been developed. The surface of the particles was functionalized with carboxyl groups ( approximately 1 COOH per 15A2) to facilitate the attachment of affinity ligands. The high surface area of the resulting beads, combined with the high density of functional groups on the surface, makes them ideally suited for the preparative purification of proteins as was demonstrated by the efficient isolation of trypsin (36 mg per gram of particles) from pancreatic extract.

Enzymatic synthesis of sorbitan esters using a low-boiling-point azeotrope as a reaction solvent.

Sorbitan esters were prepared by controlled dehydration of sorbitol followed by lipase-catalyzed esterification of the resulting "sorbitan." The reaction was carried out in azeotropic mixtures of tert-butanol/n-hexane. A partial phase diagram to determine the temperature required for the distillation of the azeotrope at a given ratio of the solvents was constructed. The effect of varying concentrations of the two solvents on the rate of esterification and the monoester/diester ratio of the final product was investigated in detail. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 351-356, 1997.

Diastereoselective resolution of 6-substituted glycosides via enzymatic hydrolysis.

The diastereoselectivity of the enzymatic hydrolyses of 4-nitrophenyl 6-deoxy-6-methyl-(R)- and (S)-sulfinyl-beta-D-galactopyranoside (1a,b), 4-nitrophenyl 7-deoxy-D- and L-glycero-beta-D-galacto-heptopyranoside (2a,b) and 4-nitrophenyl 6,7-anhydro-D- and L-glycero-beta-D-galacto-heptopyranoside (3a,b) was investigated using a range of crude glycosidase preparations. It was shown that the enzymes display a high degree of discrimination between diastereomers thereby demonstrating the utility of glycosidases for the diastereomeric resolution of unnatural 6-substituted monosaccharide derivatives.